Assessment of factors affecting in vitro shoot regeneration from axillary bud explant of Camptotheca acuminata

Axillary buds from 3-yr.-old seedlings of Camptotheca acuminata in the greenhouse were cultured on the different basal media with different concentrations of growth regulators for shoot regeneration for studying the effects of different basal media, different concen- trations of growth regulators (BA or TDZ), sucrose, agar and pH value on shoot regeneration from axillary bud. The results showed that B5 and WPM media were the optimal basal media and the optimal phyotohormone was BA of 1.0 mg/L or TDZ of 0.1mg/L; The concentrations of sucrose of 30g/L and agar of 6g/L were most suitable for the shoot regeneration; pH value from 5.8 to 6.6 were broadly effective, but the best at pH 5.8.

Regeneration of Blue Honeysuckle via Dormant Axillary Buds

The optimum medium for dormant axillary buds culture of blue honeysuckle was screened according to the growth rate and elongation rate by inoculating the buds on culture medium with various 6-BA and iron-salt concentration. About 35 days, the stretched stem buds were divided into strong root system after inoculated on 1/2 MS＋1.0 mg·L^-1 IBA rooting medium. Amount of qualified tissue-cultured young plants could be obtained by the stretched stem buds reproduction.

Rapid Propagation of Dioscorea nipponica Makino via Axillary Bud Proliferation

Objectives] This study aimed to determine the optimal medium components and culture conditions for the induction and proliferation of Dioscorea nipponica Makino axillary buds in vitro .[Methods] The effects of basic medium （MS, WPM, B5 and N6）, cytokinins type and concentration （0, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/L 6-BA; 0, 1.0, 1.5, 2.0, 2.5 and 3.0 mg/L KT）, auxins type and concentration （0, 0.25, 0.50, 0.75, 1.00 mg/L IBA, 2,4-D and NAA） on the induction of D. nipponica axillary buds were respectively measured and compared. Then, the effects of sugar source （30 g/L sucrose, fructose, white sugar, maltose and glucose） and light intensity （0, 800, 1 600, 2 400 and 3 200 lx） on the proliferation coefficient of D. nipponica axillary buds were evaluated.[Results] Using stem segments with leaf axils as the explants, the highest induction rate （90.8%） of axillary buds was achieved in MS supplemented with 2.5 mg/L 6-BA and 0.5 mg/L 2,4-D, and the fresh and dry weights of tissue culture seedlings in this medium were also the highest, up to 1.5 g and 160.9 mg, respectively. Then, the highest proliferation coefficient of was D. nipponica axillary buds noted when sucrose was used as the sugar source in medium, and the optimal light intensity for the proliferation of D. nipponica axillary buds was 2 400 lx.[Conclusions] The results provide an experimental evidence for rapid propagation of D. nipponica .

<i>In Vitro</i>Propagation of Mature Carob Trees (<i>Ceratonia siliqua</i>L.) from the Axillary Buds

The influence of tree age and the effect of growth regulators on the micropropagation of the carob (Ceratonia siliqua L.) from the axillary buds of mature trees have been described. Significant differences (P < 0.005) in results are obtained in the stages of initiation, multiplication, and rooting according to their response to the various concentrations of different growth regulators examined, namely BA, IBA, AG3. The use of 0.5 mg/l BA and 0.2 mg/l IBA was the most favorable for shoots neoformation. The leafy shoots are propagated in MS medium with BA at a concentration of 1.5 mg/l. The addition of gibberellic acid at 0.2 mg/l in the culture medium allows a good elongation and development of the shoots of the carob. The effect of the age of the plant material used has shown that explants taken from mature carob trees have a low capacity for bud sprouting and shoot proliferation compared to those taken from juvenile trees. Rooting has been successful when the plant material used is taken from young trees on an MS medium containing 2 mg/IBA, with an average number of 3 to 4, roots 1 to 2 cm long, then for the adult material, no rooting was observed. Based on these tests, it appears that micropropagation of the carob from the axillary buds is feasible, but additional work must be done to root this recalcitrant material.

Rooting of Stem Cuttings of <i>Jatropha platyphylla</i>(Euphorbiaceae) in the Obtaining of Axillary Buds for Grafting

Jatropha platyphylla is considered as a potential source of edible protein, oil, and phenolic compounds with anti-inflammatory activity. The use of stem cutting in vegetative propagation and grafting is as indispensable tools for mass multiplication of superior genotypes and helps in improve planting yield and quality. The study was aimed to investigate the effect of different diameters (10 - 15, 16 - 25 and 26 - 35 mm) and different hormone concentrations of indo-butyric acid (0, 1.5, 3, 6 and 10 g/L), in the rooting of Jatropha platyphylla and to obtain axillary buds to performed grafts. Rooting efficiency was 80% in greenhouse conditions. Hormone concentration and diameter significantly affected the rooting and shooting ability of Jatropha platyphylla stem cuttings. Stem cuttings of 26 - 35 mm have the greatest number, length and dry root weight. 76% survival of the grafted plants was obtained. This demonstrates the necessity to improve the conventional propagation methods with appropriate scientific inputs for more economical and time efficient techniques for standard propagation protocols.

In vitro Axillary Bud Multiplication of Citrus Nobilis Lour. in Indonesia

This research aimed to get the best composition ofcytokinin （BAP） and auxin （NAA） concentration on in vitro axillary bud multiplication of Tawangmangu orange （Citrus nobilis Lour.）. The research was conducted in Plant Physiology and Biotechnology Laboratory of Agriculture Faculty, Sebelas Maret University Surakarta from February 2008 to September 2008, used factorial design with two factors based on Completely Random Design. The first factor was BAP concentration consisting of five levels： 0, 0.5, 1, 1.5, 2 ppm and the second factor was NAA concentration consisting of five levels： 0, 0.5, 1, 1.5, 2 ppm. So there were 25 combination treatments, each of them was repeated for three times. The result observation data of shoot initiation time and number of shoot were analyzed with analysis of variance and if significant continued with Duncan＇s Multiple Range Test at 5% level. Data of shoot color and amount of leaves were analyzed descriptively. The result showed that there was interaction between BAP and NAA on shoot initiation time. Combination of 0.5 ppm BAP and 1.5 ppm NAA was able to accelerate the shoot initiation time and produced the largest number of leaves that was eight leaves. BAP was able to increase the number of shoots. Average largest amount of shoot on 0.5 ppm BAP treatment was 3.93 shoots/explant. The dominant color of leaves was light green.

Conservation of an important montane bamboo Thamnocalamus falconeri, Hook.f.ex Munro through axillary bud proliferation

Thamnocalamus falconeri, Hook.f. ex Munro.,an important bamboo species belonging to the family Poaceae, locally known as Ringal, occurs in the hills of Uttarakhand, India. This species has been traditionally exploited by local communities to support their livelihoods.Increasing needs of the hill villages impose unsustainable pressure on natural stands of Ringal in the Uttarakhand hills and forests have been degraded. The long history of excessive cutting of Ringal from natural forests and the lack of replanting threaten villager livelihoods. Replanting is required to conserve the species. We propose a protocol for generation of planting material through axillary bud proliferation for multiplication and conservation of this species. We collected offsets/rhizomes from a natural stand of T. falconeri in the Chopta Mandal areas（Chamoli district, India）. These were planted at sites of varied elevation and fresh single nodal segments were collected from them as explants. Different sterilization treatments were assessed to combat contamination. Among these, treatment of 0.1 %Hg Cl2 followed by 5 % Na OCl, proved best. Among two cytokinin treatments, viz. BAP and Kinetin, singly or in combination, BAP alone（5 mg L-1） proved superior and resulted in 100 % bud break. BAP-supplemented MS media yielded maximum vigorous shoot formation（90 %）and maximum number of shoots（8.9）. Subculturing of shoots on the same medium with similar BAP treatment（5 mg L-1BAP） enabled continuous production of healthy shoots at similar frequency. Maximum rooting（100 %）was recorded on half-strength MS medium supplemented with 5 mg L-1IBA. Micropropagated plants were hardened and acclimatized in soil mixture（2：1：1） and then transplanted to field sites（Magra, Uttarakhand, 1,834 m）.Eight to ten months after field transplantation we recorded100 % survival of transplanted material. This micropropagation protocol could be used successfully for raising a stock of genetically homogenous plant material in bulk for field plantations and for conservation of the species.

Non-dormant Axillary Bud 1 regulates axillary bud outgrowth in sorghum

Tillering contributes to grain yield and plant architecture and therefore is an agronomically important trait in sorghum （Sorghum bicolor）. Here, we identified and functionally characterized a mutant of the Non- dormant Axillary Bud 1 （NAB1） gene from an ethyl methanesulfonate-mutagenized sorghum population. The nab1 mutants have increased tillering and reduced plant height. Map-based cloning revealed that NAB~ encodes a carotenoid-cleavage dioxygenase 7 （CCDT） orthologous to rice COryza sativa） HIGH-TILLERING DWARF1/ DWARF^7 and Arabidopsis thaliana MORE AXILLARY BRANCHING 3. NAB1 is primarily expressed in axillary nodes and tiller bases and NAB1 localizes to chloroplasts. The nab1 mutation causes outgrowth of basal axillary buds; removing these non-dormant basal axillary buds restoredthe wild-type phenotype. The tillering of nab1 plants was completely suppressed by exogenous application of the synthetic strigolactone analog GR24. Moreover, the nab1 plants had no detectable strigolactones and displayed stronger polar auxin transport than wild-type plants. Finally, RNA-seq showed that the expression of genes involved in multiple processes, including auxin-related genes, was significantly altered in nab1. These results suggest that NAB1 functions in strigolactone biosynthesis and the regulation of shoot branching via an interaction with auxin transport.

Brassinosteroid Biosynthetic Gene CmDWF4 Regulates Bud Outgrowth in Chrysanthemum morifolium

Brassinosteroids(BRs),a class of steroid phytohormones,play a critical role in plant growth and development.The DWF4 gene encodes a cytochrome P450 enzyme(CYP90B1),which is considered a rate-limiting enzyme in BR biosynthesis.Here,we identified a homologous gene of DWF4 in chrysanthemum,CmDWF4.This gene was predicted to encode 491 amino acid residues with a molecular weight of 56.2 kDa and an isoelectric point(pI)of 9.10.Overexpression of CmDWF4 in chrysanthemum was found to significantly increase growth rate,number,and length of lateral buds.Transcriptome analysis showed that multiple xyloglucan endotransglycosylase/hydrolase(XTH)family encoding genes associated with cell wall modification were up-regulated in CmDWF4-overexpressing lines.qRT-PCR assay confirmed the up-regulation of CmXTH6,CmXTH23,and CmXTH28 in CmDWF4-overexpression line.Overall,this work establishes a mechanism by which BR biosynthetic gene CmDWF4 promotes lateral bud outgrowth in chrysanthemum,possibly through regulating cell elongation and expansion.

In Vitro Propagation, Isolation and Expression Studies of Suaeda edulis Genes Involved in the Osmoprotectants Biosynthesis

Halophytes are an excellent choice for the study of genes conferring salt tolerance to salt-sensitive plants and,they are suitable for reclamation and remediation of saline soil.We develop an in vitro plant propagation protocol and studies of genes involved with GB and Pro biosynthesis in Suaeda edulis.Axillary buds were used as explants and cultured in different treatments on Murashige and Skoog(MS)medium supplemented with different concentrations and combinations of plant growth regulators.The highest number of multiple shoots was on MS medium containing 1 mg/L Benzyladenine(BA)and/or 2 g/L activated carbon with 5.5±06 shoots per explant.The identification and expression analysis of genes involved in glycine betaine(GB)biosynthesis were S-adenosyl-methionine synthetase(SAMS),choline monooxygenase(CMO)and betaine alde-hyde dehydrogenase(BADH),and for proline(Pro)was pyrroline 5-carboxylate synthetase(P5CS).These sequences shared 90–95%of identity with others plant homologous in public databases.The amino acids sequence analysis showed that all these peptides contain some of the conserved motifs of those kinds of enzymes.The qRT-PCR analysis revealed a higher expression of SeBADH,SeCMO,and,SeP5CS genes in the roots and leaves from plants collected in the field in contrast with from in vitro plants.However,the expression level of SeSAMS was higher only in the leaves of plants collected in the field when com-pared to those cultivated in vitro.

Genetic Stability of Cryopreserved Grapevine （Wtis vinifera L.） Genome by Vitrification Method

Grapevine （Vitis spp.） is an economically important fruit crop worldwide. In Mexico, Sonora State leads the table grape production and exportation to international markets. In this regard, it is important to preserve the grape varieties during long time without phenotypical or genotypical changes. Cryopreservation is a good alternative, although it very often can induce changes in genome and phenotype. In this study, grapevine cv. ＂Flame Seedless＂ axillary buds were cryoprcserved by vitrification using the plant vitrification solution 2 （PVS2） and stored in liquid nitrogen （LN） for one hour, one week and one month, respectively. Genetic stability of buds cryopreserved under all treatments was evaluated using inter-simple sequence repeats （ISSR） markers. Ten ISSR primers were evaluated, but only two primers were possible to amplify distinct and reproducible bands with sizes between 300 bp and 2,000 bp. Different ISSR fragment patterns were recorded in cryopreserved buds as compared with control. These results suggest that cryopreservation by LN and vitrification-cryopreservation affect genetic stability in grapevine.

Establishment of Regeneration System of Bougainvillea ‘Yunnan Purple’ Stem Segment

To optimize the regeneration system of Bougainvillea, the effects of different hormone ratios and concentrations on axillary bud induction, proliferation and re-rooting were studied using annual semi-lignified branch cuttings of Bougainvillea ‘Yunnan Purple’ as experimental materials. The results showed that MS+6-BA 2.5 mg/L+NAA 0.1 mg/L was the optimal medium for stem axillary bud initiation, and the initiation rate reached 91.3%. The optimal medium for axillary bud proliferation was MS+6-BA 1.0 mg/L+NAA 0.2 mg/L, and the proliferation coefficient was 3.28. The optimal rooting medium was 1/2 MS+IBA 1.0 mg/L, with the rooting rate of 90% and the average root number of 7.4. After 15 d of hardening seedling, the survival rate of the sterile seedlings was 97.83%. This study laid a basis for rapid propagation and genetic transformation system of Bougainvillea.

In Vitro Propagation of Echeveria elegans, a Species of the Flora Endangered Mexican

The genus Echeveria has about 143 species that are unique to the Americas, 117 of which are presented in the Mexican Republic, mainly in the states of Hidalgo, Puebla and Oaxaca. Propagation can be done by cuttings, leaf cuttings or seeds, but these methods are insufficient to avoid the predation of many species. NOM-059-ECOL-2001 shows Echeveria elegans as species reported endangered. The objectives in this study were achieved in vitro propagation from axillary buds, as an alternative to multiplication and preservation species. We evaluated different concentrations and combinations of two growth regulators, 6-BAP （2.22, 4.44, 6.66 and 8.88 μM） and u-NAA （1.35 and 2.70 μM） on shoot formation from axillary buds and two culture media with different concentrations of MS salts, to achieve root plants grown in vitro. It was found by combining 6.66 μM of 6-BAP and 1.35μM of α-NAA favored the formation and development of new shoots. In the rooting stage, the best results are achieved with the treatment containing 50% of MS salts. Then the plants were transplanted into greenhouses and achieved a successful acclimatization. During this last phase of development, there were no phenotypic changes or presence of somaclonal variants.

Nicotine Concentration in Leaves of Flue-cured Tobacco Plants as Affected by Removal of the Shoot Apex and Lateral Buds

It is believed that the nicotine concentration in tobacco is closely correlated with the amount of nitrogen （N） supplied. On the other hand, N uptake mainly occurs at the early growth stage, whereas nicotine concentration increases at the late growth stage, especially after removing the shoot apex. To identify the causes of the increased nicotine concentration in tobacco plants, and to compare the effects of different ways of mechanical wounding on nicotine concentration, field experiments were carried out in Fuzhou, Fujian Province in 2003 and 2004. Excision of the shoot apex had almost no influence on N content in the plant; however, it caused dramatic increases in nicotine concentration in leaves, especially in the middle and upper leaves. An additional increase of the nicotine concentration was obtained by removal of axillary buds. The wounding caused by routine leaf harvests, however, did not change the leaf nicotine concentration, and neither did reducing leaf harvest times. The present results revealed no direct relationship between N supply and nicotine concentration in tobacco leaves, and indicate that not all kinds of mechanical wounding were capable of stimulating nicotine synthesis in tobacco plants. Since nicotine production is highly dependent on the removal of apical meristems and hence on the major sources of auxin in the plant, and application of 1-naphthylacetic acid onto the cut surface of the stem after removing the shoot apex markedly decreased the nicotine concentration in different leaves and the total nicotine content in the plant, the results suggest that decreased auxin supply caused by removal of the shoot apex as a kind of mechanical wounding might regulate nicotine synthesis in the roots of tobacco plants.

Bioinformatics and Expression Analysis of CPMAX2 in Citrange

Transgenic citrange lines with rol ABC genes behave rosette branching and extreme dwarfing.To explore the regulatory mechanism of plant hormones in axillary shoot growth of transgenic citrange,a full length cD NA of CPMAX2 was cloned from citrange [Citrus sinensis(L.) Osb × Poncirus trifoliate(L.) Raf.] by RT-PCR in this study.The expression of CPMAX2 was detected in axillary tender leaves of 3 transgenic citrange lines and the wild type.Additionally,we constructed an over-expression vector CPMAX2-p CAMBIA1301 for further study.The results showed that the c DNA sequence and its putative peptide sequence shared 99.66% and 99.14% of identity with its Citrus sinensis ortholog MAX2.The deduced amino acid sequence contained putatively one F-box and two LRR repeat domains that are highly conserved in MAX2 genes.CPMAX2 was obviously down-expressed in tender leaves of 3 transgenic citrange lines compared with the wild type.The results suggest CPMAX2 play an important role in regulating the rosette shoot growth in transgenic citrange with rol ABC genes.